GEF-H1/RhoA信号通路在肝素结合蛋白诱导严重烧伤早期血管内皮细胞损伤中的作用

In the early severe burns, increasing level of Heparin-binding protein (HBP) in plasma is closely associated with expression of cytokines and dysfunction of systemic vascular permeability. Our previous studies confirmed that GEF-H1/RhoA signaling pathway mediated endotoxin-induced expression of inflammatory cytokines and permeability dysfunction in endothelial cells. In the present study, we found HBP not only could be derived from the neutrophil, but also from vascular endothelial cells. HBP not only increased significantly expression of GEF-H1, but also upregulated significantly levels of p-MYTP-1, p-p65 and IL-6 mRNA in endothelial cells. Taken together, we assume that that HBP regulates expression of inflammatory cytokines and vascular permeability compromise through activation of GEF-H1/RhoA signaling pathway in the early severe burns.The purpose of study is to fully illustrate the mechanisms of systemic edema formation, shock and SIRS during the early stage after severe burns, and to provide strategical methods for clinical tratment.

严重烧伤早期，血浆肝素结合蛋白（HBP）的水平已证实与炎症因子产生和毛细血管通透性的增加等密切相关。我们前期研究结果显示：GEF-H1/RhoA信号通路的活化是血管内皮细胞炎症因子的产生与释放以及内皮细胞通透性增加的重要因素之一。本项目的预实验结果提示内毒素等外界刺激能诱导血管内皮细胞高表达HBP，采用HBP 作用于血管内皮细胞, 不仅细胞内GEF-H1表达显著增加，而且MYPT-1和p65的磷酸水平以及IL-6 mRNA 的表达均明显升高。因此，我们推测严重烧伤早期，HBP通过激活内皮细胞GEF-H1/RhoA信号通路，介导炎症因子的表达和内皮细胞通透性的改变。本项目采用机制研究和治疗干预手段相结合，通过研究HBP对血管内皮细胞的具体作用机制，从而更加全面地理解严重烧伤早期全身水肿形成、休克及SIRS产生的细胞和分子生物学机制，为临床防治提供新的思路。

此项课题中我们着力于内皮细胞中RhoA/ROCK 信号通路的研究。内皮细胞收缩能够导致内皮细胞屏障破坏引起血管内皮细胞间高通透性改变；内皮细胞收缩依赖于细胞内骨架蛋白改变及肌动蛋白纤维束形成产生的向心力。而RhoA/ROCK 信号通路活化是产生这种向心力所必须。 RhoA/ROCK 能够直接催化MLC磷酸化或者间接失活MYPT1引起MLC磷酸化水平升高， MLC磷酸化（p-MLC）及MYPT1磷酸化（p-MYPT1）水平可以用来做为RhoA/ROCK活化致内皮细胞肌动蛋白骨架重构、应力纤维形成，进而引起内皮细胞收缩的可靠参考指标。GEF-H1激活后能够将LPS刺激信号传导至RhoA/ROCK、磷酸化下游效应分子MLC和MYPT1，引起肌动肌球蛋白交联增加、细胞内肌动蛋白骨架重建和应力纤维生成，最终导致内皮细胞收缩，破坏内皮细胞屏障功能。此项研究不仅从分子和细胞学水平上了解炎症状态下内皮细胞的生物学特性，且对脓毒症休克的临床诊断治疗意义重大。